Project Summary In the US, 20,000 cases of oral cavity cancer are diagnosed annually, of which the majority are tongue squamous cell carcinoma. Five-year survival for oral cancer, at 54% has not improved in the past 50 years. It is one of the lowest of the major cancer sites, resulting in more people dying from oral cancer than melanoma or ovarian cancer. Development of oral cancer is considered a multistep process involving precancerous lesions (oral epithelial dysplasia) of increasing severity. Transformation to oral cancer increases with grade. Once invasive cancer develops, neck (cervical) metastasis poses the greatest risk to patient survival. Currently, there are no clinical, pathologic or molecular markers to discriminate dysplasias that will progress or cancers with aggressive potential (i.e., risk of metastasis). Progression to oral cancer is associated with acquisition of epigenetic and genomic alterations, including amplification of focal regions of the genome. Changes in the metagenome also accompany oral cancer. In an initial study, we reported that the cancer-associated microbiome is characterized by reduced abundance of early colonizing bacterial genera, Streptococcus, Actinomyces and Rothia and increased abundance of Fusobacterium. A subsequent, larger metagenomic and genomic study of 50 patients confirms the cancer associated dysbiosis and identifies a group of node positive (N+) tongue cancers in which high abundance of Fusobacterium and amplification of chromosome 11q13.3 and/or 11q22 co-occur. These two amplicons together encompass a dozen putative oncogenes (e.g., CCND1, YAP1). We hypothesize that overexpression of genes in the 11q amplicons cooperates with Fusobacterium to promote N+ tongue cancer. Moreover, since amplification of 11q13.3 and 11q22 are present in precancers, we propose that cooperation of genes in the 11q amplicons with Fusobacterium could be an early event, initiating a developmental pathway culminating in aggressive oral cancers (i.e., those with potential for invasion and lymph node metastasis). Here, we will validate our initial findings of N+ tongue cancers with high abundance of Fusobacterium and amplification of 11q13.3 and/or 11q22 by genomic and metagenomic analysis of an independent cohort and determine the status of 11q amplification and abundance of Fusobacterium in dysplasia associated with these cancers (Aim 1). In Aim 2, we will determine whether F. nucleatum promotes carcinogenesis in a mouse model and if transformation is associated with stereotyped genome alterations. Successful completion of Aim 1 offers the exciting possibility that 11q amplification and abundance of Fusobacterium can be developed into a biomarker identifying N+ tongue cancer patients, as well as precancer patients at risk for progression to aggressive disease. Aim 2 provides a foundation for future studies leading to a mechanistic understanding of the interaction between genes in the 11q amplicons and Fusobacterium.